WO2007113188A1 - Précipitation biphasique de protéines - Google Patents

Précipitation biphasique de protéines Download PDF

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Publication number
WO2007113188A1
WO2007113188A1 PCT/EP2007/052976 EP2007052976W WO2007113188A1 WO 2007113188 A1 WO2007113188 A1 WO 2007113188A1 EP 2007052976 W EP2007052976 W EP 2007052976W WO 2007113188 A1 WO2007113188 A1 WO 2007113188A1
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WO
WIPO (PCT)
Prior art keywords
protein
solution
salt
proteins
organic compound
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Application number
PCT/EP2007/052976
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English (en)
Inventor
Thorkild Christensen
Jan Anton Enoksen
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Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to EP07727448.8A priority Critical patent/EP2004671B1/fr
Priority to US12/294,278 priority patent/US8088900B2/en
Publication of WO2007113188A1 publication Critical patent/WO2007113188A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation

Definitions

  • Precipitation is a widely used method for the recovery of proteins from various solutions. Proteins in solution vary greatly in their solubility depending on their amino acid composition and the characteristics of the solvent (such as ionic strength and temperature). A number of methods for precipitation of proteins are well known. Protein crystallization, for example, is frequently used to precipitate proteins, but this method requires typically comprehensive development to achieve right crystallization conditions. lsoprecipitation is another frequently used protein precipitation method, making use of the fact that a proteins solubility has a minimum at the protein's isoelectric point (Pl). However, due to posttranslational modifications a number of proteins do not have a well defined Pl.
  • Pl isoelectric point
  • coagulation factor VII (FVN” or “FVIIa” (when referring to the activated form of FVII)) comprises y-carboxy groups and sialic acid groups, but not in any fixed number, making the pi of FVII difficult to define.
  • Protein precipitation also frequently is induced by addition of an organic solvent, to alter the nature of the solution in which the protein is contained.
  • an organic solvent to alter the nature of the solution in which the protein is contained.
  • dehydration of protein surfaces is a reason (if not the reason) for precipitation of proteins in the presence of organic solvents.
  • Salting out protein precipitation methods have been used extensively, particularly before the development of more recently developed chromatographic purification methods. Salting out of proteins makes use of the fact that a protein's solubility often decreases at high salt concentrations (and proteins can therefore be made to precipitate under such conditions). Various salts have different salting out effects, but the general principles for salting out are presented in the so-called Hofmeister series that have been known for more than 100 years. Besides the particular salt used, the nature of the protein in question also influences its ability to be precipitated by such a method. Ammonium sulphate is among the salts that most often has been used for precipitation of proteins. The methods of salting out precipitation, organic solvent precipitation, and isoprecipitation, have frequently been used in isolation, rather than in combination with each other.
  • TPP Three Phase Partitioning
  • TPP Three Phase Partitioning
  • EP0474391 discloses a method in which an acylated protein (insulin) may be isolated by adjusting the pH to a value near the pi of the protein. Then a suitable amount of an alcohol is added. In the example ethanol is used in an amount of 0.46 liter per liter solution to give a precipitation, in fact a so-called isoprecipitation is described. No salt is added.
  • EP1561756 discloses the removal of impurities like DNA and viruses from a protein solution (particularly antibodies). This is done by lowering the pH to a value equal to or lower than the pi of the protein with low conductivity. Under these conditions impurities as particles may be removed, while the protein of interest remains in solution. No organic solvent is used.
  • Figure 1 illustrates the results obtained in precipitating IL21 in NaCI or (NH4)2SO4 with benzyl alcohol according to one aspect of the invention.
  • Figure 2 illustrates the results obtained in precipitating hGH in NaCI or ammonium sulphate buffers according to another aspect of the invention.
  • Figure 3 illustrates the results obtained in precipitating rFVIIa according to another aspect of the invention.
  • Figure 4 illustrates the results obtained in precipitating interleukin-21 with butanols according to another aspect of the invention.
  • the invention described herein provides a method of precipitating a protein comprising (a) providing a protein solution, (b) adding a salt to the solution, (c) adjusting pH of the composition to below the pi of the protein, and (d) adding an organic compound to the solution, so as to form a two-phase system, wherein the protein is precipitated in a solid protein phase, which may then for instance be separated from the liquid phase by filtration.
  • a protein solution can be provided by any suitable means and the inventive method can be practiced with any suitable type of protein solution.
  • a protein solution can be, for example, the product of recombinant protein expression; a biological sample taken from an organism; or a solution containing a protein produced by chemical synthesis methods.
  • a protein solution may also be provided as purification product of such a medium, such as a fraction from a chromatography purification step.
  • protein is used herein as referring to peptides (single chain amino acid polymers of about 3-50, typically about 3-40 amino acid residues in length) as well as polypeptides (single chain amino acid polymers of 50 and more amino acid residues) and more complicated (e.g., multi-chain) proteins.
  • the protein solution can include any number of proteins of interest and/or undesirable ("contaminating") proteins.
  • the solution may include one, two, three, or more proteins of interest.
  • the target protein is a protein that is subjected to one or more variable posttranslational modifications, that cause the protein to be associated with a poorly defined Pl.
  • coagulation factor VII proteins and analogues or derivatives of such proteins having similar features in terms of posttranslational modifications are an example of such a protein.
  • proteins that the method can be advantageously applied to include growth hormone proteins (GHs) and interleukins (“ILs”), such as IL-2, IL-20, and IL- 21.
  • Other proteins of interest may include insulins; glucagon-like proteins (e.g., GLP-1 or GLP-2); other coagulation factors (e.g., factor VIII, factor IX, factor Xl, or factor XIII); erythropoietin; vascular endothelial growth factors (VEGFs); interferons (e.g., IFN- ⁇ , IFN- ⁇ , or IFN- ⁇ ), colony stimulating factors (e.g., G-CSF); soluble receptor proteins (e.g., soluble TNF ⁇ receptor proteins); tissue plasminogen activator (tPA); antibodies; IGF-1 ; fibroblast growth factor (FGF); and analogues of such proteins, derivatives of such proteins, and fusion proteins related to such proteins (e.g., albumin-interferon fusion proteins).
  • At least about 75% of the protein is precipitated in the solid protein phase.
  • the two-phase system contains a solid protein phase and no more than one liquid phase.
  • the salt added to the solution can be any suitable salt.
  • Suitability in the context of the inventive method means that the salt is capable to induce the effects necessary, in combination with the other steps of the method, to achieve a suitable level of precipitation with respect to the protein(s) of interest (or "target proteins," which may be, e.g., the entire protein content of the composition).
  • Suitability also typically means that the salt does not include any ions that may be detrimental to the health of someone receiving a protein solution comprising the salt, such as wherein the method is applied to concentrate a protein for purposes of generating a pharmaceutical composition.
  • the salt added to the solution typically is a so-called “salting-out” salt.
  • salting out salts include sulphate salts, chloride salts, nitrate salts, phosphate salts, citrate salts, and thiocyanate salts. Specific examples of suitable salts are known in the art.
  • the salt is a "salting-in” salt. Additional examples of suitable "salting-in” salts are known.
  • the salt is selected from NaCI or ammonium sulfate.
  • Ammonium sulfate is very soluble, allowing for a range of concentrations to be applied to precipitate a variety of proteins under different conditions (which is particularly advantageous in other aspects of the invention wherein precipitation of the target protein is not desired).
  • ammonium sulfate is typically an advantageous salt in the practice of the inventive methods described herein.
  • Other suitable salts include ammonium acetate, citric acid salts, and tartaric acid salts.
  • the salt can be added in any suitable concentration. Suitability with respect to concentration also refers to effectively obtaining precipitation of the desired protein product or achieving other desired effect.
  • concentration also refers to effectively obtaining precipitation of the desired protein product or achieving other desired effect.
  • the ordinarily skilled artisan will recognize that different proteins may become less soluble (tends towards “salting out") under different conditions, such that the amount of salt that will be most effective will vary (but the appropriate amount can be readily determined using routine experimentation).
  • An advantage of the method is that the amount of salt used in the precipitation process typically is significantly less than an amount used in a traditional "salting out” precipitation (thereby, among other things, resulting in less risk of protein denaturation or less amounts of undesirable denatured protein products than obtained in such methods).
  • the final salt concentration achieved in step (a) of the method is in the order of about 0.1 to about 3 M (e.g., about 0.2 M to about 2 M, about 0.05M to about 1 M, for example about 0.2-0.8 about M, such as 0.2-0.6 about M, such as about 0.2-0.4 M, e.g., about 0.25 M).
  • the pH of the composition is lowered to some suitable amount below the pi of the protein.
  • the lowering of the solution can be achieved by any suitable method.
  • an acid such as sulfuric acid, is added to the solution so as to lower the pH to the desired level.
  • suitability is associated with the desired precipitation effect.
  • the pH obtained in this step should be about 0.5 to about 5 pH units below the protein's Pl, such as about 1-4 units below the protein's Pl, such as about 1.5-3.5 units below the protein's Pl, for example about 2 units below the protein's Pl.
  • the organic compound is water soluble.
  • the organic compound is hydrophobic and water soluble.
  • solvents include benzyl alcohol, n-butanol, and benzoic acid. In general, phenol and phenol derivatives may be suitable. Benzyl alcohol has been found to be particularly advantageous in the practice of the method.
  • the organic compound is t-butanol. However, in another aspect, the organic compound is defined as not being t-butanol.
  • the water soluble organic solvent is not a polymer.
  • the organic solvent is added in an amount, which between 0.5% and 5% of the resulting solution In one embodiment, the organic solvent is added in an amount, which is not more than about 5%, such as about 5% of the resulting solution. In one embodiment, the organic solvent is added in an amount, which is not more than about 4%, such as about 4% of the resulting solution. In one embodiment, the organic solvent is added in an amount, which is not more than about 3%, such as about 3% of the resulting solution.
  • the organic solvent is added in an amount, which is not more than about 2%, such as about 2% of the resulting solution. In one embodiment, the organic solvent is added in an amount, which is not more than about 1 %, such as about 1 % of the resulting solution.
  • the organic solvent that is selected has a protein preservative effect.
  • benzyl alcohol and benzoic acid are both known to be preservatives.
  • the precipitated protein product also is protected against undesirable microbial growth.
  • the amount of organic solvent used in generally low (less than about 10%, and typically only a few percent).
  • the amount may be, e.g., about 0.25 to about 4%, such as about 0.5 to about 3%, such as about 0.75-2.5%, for example about 1 % to about 2%.
  • These amounts are significantly less than used in, e.g., a TPP method.
  • the protein solution is gently stirred.
  • the method can generally be practiced at room temperature conditions.
  • the addition of the organic solvent creates a protein-rich solid phase and a protein- poor phase in the solution.
  • the two phase system lends itself well to further purification of the protein by conventional methods, such as filtration and/or centrifugation techniques.
  • target protein precipitation yields as high as about 80% or more, about 85% or more, about 90% or more, or even 95% or more (e.g., nearly 100% or 100% (within means of detection)) may be achieved.
  • the precipitated product may be kept in solution for considerable lengths of time advantageously in association with the prevention of certain types of degradation often associated with proteins in solution, such as dimerization, polymerization, or generation of desamido forms. Proteins in solid form may resist such processes and otherwise be more stable than proteins in solution. Precipitated proteins also may be able to better retain proper tertiary structure over lengths of time.
  • one aspect of the invention involves the generation of a precipitated protein product and the use of that product as an intermediate for the later generation of a purified protein solution.
  • the intermediate or purified product is subjected to freezing or freeze-drying.
  • the precipitation step also can serve as a method of concentrating protein(s) in the solution or target proteins in the solution (either by precipitation of such proteins or, in the aspects described below by retaining such proteins in solution while precipitating "contaminating" (undesirable) protein species in the protein solution).
  • the method can serve as a method of concentrating a protein.
  • the invention provides a method of purifying a protein solution comprising (a) providing a protein solution, (b) adding a salt to the solution, (c) adjusting pH of the composition to above the pi of the protein, and (d) adding an organic compound to the solution so as to obtain precipitation of protein impurities from the solution, and removing precipitated protein impurities from the protein solution.
  • the basic difference in the method of this aspect is that the conditions used are such that complete protein precipitation is avoided and that precipitation of undesirable proteins is obtained while desirable protein(s) are kept in solution. This can be accomplished by, e.g., modifying the amount of ammonium sulphate used in the salt addition step of the method.
  • MW proteins For example, below 25% ammonium sulphate saturation, particulate or high molecular weight (MW) proteins precipitate. Further sequential “cuts” can also be obtained by increasing the concentration in 10-20% incremental steps until proteins that are desired to be precipitated are precipitated - where total precipitation is desired an amount such as about 80-90% can be used.
  • the salt and pH adjustment steps also can be modified to retain desirable proteins in solution - such as, e.g., not reducing the pH significantly below the pi of target proteins.
  • the method of this aspect can be used to purify solutions containing proteins of interest, such as, e.g., where the target protein is a recombinant product (e.g., hGH, FVII, FXIII, FXI, an insulin, etc.) and the initial protein solution is a lysate from a recombinant cell that expressed that product (e.g., a recombinant hGH-expressing bacterial cell lysate) or other type of complex protein mixture (e.g., a biological sample comprising many types of proteins in addition to the target protein).
  • the pH may be increased to above the target protein's Pl, and the other steps performed so as to remove impurities while keeping the target protein in solution.
  • this second aspect method (wherein the target protein is kept in solution) may be combined with the first aspect (wherein the target protein is precipitated) as a combined method for removing undesirable proteins and concentrating desired ones.
  • the various methods of the invention can be combined with other protein purification methods such as chromatographic purification, filtration, etc.
  • the practice of the methods can be characterized as lacking the performance of these or other steps (e.g., in one aspect, the method is characterized by the lack of a crystallization step, the lack of any precipitation-inducing polymer, etc.).
  • the solution is free of mannitol or other agents that may be found in a final protein solution.
  • the concentration in the IL21 mixtures was approximately 1.3 mg IL21/ml in 2 M NaCI and the concentration of benzyl alcohol varied from 0-4%.
  • the mixtures were prepared and left for a short while followed by filtration. UV absorption of the supernatants was measured at A 280 and the absorbance measurements converted to total amount of mg in the supernatants. The result is shown in Fig 1 - upper curve (blue).
  • the concentration in the mixtures of IL21 here was 1.1 mg IL21/ml in 1.5 M AMS and benzyl alcohol content varied from 0-4%.
  • the mixtures were prepared and left for a short while followed by filtration. UV absorption of the supernatants was measured at A 280 and the absorbance measurements converted to total amount of mg in the supernatants. The result is shown in Fig 1 - lower curve (purple).
  • FVIIa is subject to post translational modifications - i.e., both ⁇ -carboxylated and glycosylated also with sialic acid residues, it does not have a well defined pi (rather FVIIa can be characterized in association with a pi range). However, the modifications will lower pi as compared with an unmodified form of the protein.
  • Example 5 Precipitation with n-butanol and isobutanol in NaCI and AMS solutions.
  • IL21 is not precipitated with butanol in NaCI solutions. However, in 1.5 M AMS IL21 is precipitated. With 4% n-butanol about 83% IL21 is precipitated and about 60% with iso-butanol.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne de nouveaux procédés de précipitation de protéines, les procédés consistant à (a) obtenir une solution protéique, (b) ajouter un sel à la solution, (c) soit (i) ajuster le pH de la composition à une valeur inférieure au pI de la protéine (dans les cas où le procédé porte sur la précipitation de la plupart ou de la totalité des protéines présentes dans la solution), soit (ii) ajuster le pH de la composition à une valeur supérieure au pI de la protéine (dans les cas où le maintien de la protéine cible dans la solution est souhaité), et (d) ajouter un composé organique à la solution. En outre, (I) lorsque le procédé implique l'étape [(c)(i)], il se forme une solution biphasique, au moins 75 % environ de la protéine étant contenus dans la phase protéique, et (II) lorsque le procédé implique l'étape [(c)(ii)], ledit procédé consiste en plus à éliminer les impuretés précipitées de la solution protéique.
PCT/EP2007/052976 2006-03-30 2007-03-28 Précipitation biphasique de protéines WO2007113188A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP07727448.8A EP2004671B1 (fr) 2006-03-30 2007-03-28 Deux phases précipitation des protèines
US12/294,278 US8088900B2 (en) 2006-03-30 2007-03-28 Two-phase precipitation of proteins

Applications Claiming Priority (2)

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EP06111966 2006-03-30
EP06111966.5 2006-03-30

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WO2007113188A1 true WO2007113188A1 (fr) 2007-10-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8088900B2 (en) * 2006-03-30 2012-01-03 Novo Nordisk A/S Two-phase precipitation of proteins

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CN110462400B (zh) 2017-03-28 2022-06-21 相达生物科技国际有限公司 用于在液体活检中准确诊断疾病靶向生物标记的方法
CN110891664B (zh) 2017-06-01 2022-05-17 相达生物科技美国有限公司 用于多孔材料中双水相分离的相分离行为改性剂
US11609232B2 (en) 2017-09-01 2023-03-21 Phase Diagnostics, Inc. Method and device of using aqueous two-phase systems (ATPS) for enhancing diagnostics for sexually transmitted infections
US12043826B2 (en) 2018-01-19 2024-07-23 Phase Scientific International, Ltd. Spontaneous nucleic acid purification and concentration in a single step
EP3740588A4 (fr) 2018-01-19 2021-10-20 Phase Scientific International, Ltd. Procédé d'isolement et de purification d'acides nucléiques à l'aide d'un système à phases solide-liquide
WO2019143943A2 (fr) 2018-01-19 2019-07-25 Yin To Chiu Composition et procédé de concentration et d'enrichissement d'acides nucléiques
WO2019144016A1 (fr) 2018-01-19 2019-07-25 Yin To Chiu Méthode d'isolement d'exosomes à l'aide d'une encapsulation et système micellaire aqueux

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WO1997034919A1 (fr) * 1996-03-15 1997-09-25 Novo Nordisk A/S Procede de purification d'une proteine presente dans une solution contenant des proteines
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US5728559A (en) * 1993-07-09 1998-03-17 Novo Nordisk A/S Separation of proteins
EP0747391A2 (fr) * 1995-06-07 1996-12-11 Eli Lilly And Company Procédé de fabrication d'une poudre de protéines acyles
WO1997034919A1 (fr) * 1996-03-15 1997-09-25 Novo Nordisk A/S Procede de purification d'une proteine presente dans une solution contenant des proteines
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Publication number Priority date Publication date Assignee Title
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US20090286966A1 (en) 2009-11-19
US8088900B2 (en) 2012-01-03
EP2004671A1 (fr) 2008-12-24
EP2004671B1 (fr) 2015-12-09

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